Process for monohydroxylation of steroids



in the water that is used in the process. ever, it is advisable tosupplement the minerals normally H '-present with added amounts toobtain maximum growth" United States Patent PROCESS FORMONOHYDROXYLATION OF STEROIDS No Drawing. Filed July 17, 1959, Ser. No.827,698

11 Claims. Cl. 195-51 This invention relates to the monohydroxylation ofsteroids. More particularly, this invention relates to themicrobiological monohydroxylation of steroids of the pregnene series bythe action of fungi of the class Basidiomycetes. p

The microbiological fermentation of steroids to producemonohydroxylation is well known. In US. Patent No. 2,789,940 amicrobiological process for the conversion of Reichsteins Substance S tohydrocortisone is described and claimed. However, there is nomicrobiological process heretofore described which will produce as majorproducts three monohydroxylated steroids of the pregnene series asdescribed hereinafter. I

The process of the present invention is a fermentative oxidation usingfungi of the class Basidiomycetes. The genuses Coriolus, Polyporus andPoria are particularly useful in the present process; for example,Coriolizs versicolor (ATCC No. tulipiferus (ATCC No. 13,489) (D-l5),Polyporus cinnabarinus (D-6) or Poria cocos (ATCC No. 13,490) (D-107). Adescription of the fungus Coriolus versicolor is given by F. L. Stevens(Plant Disease Fungi, page 304, Macmillan Co., New' York, 1942) underthe former generic name Polystictus. Polyporus tulipiferus is describedby Darley and Christensen in Phytopathology 35 220222 (1945). The fungusPoria COCOS is described by Weber in Mycologia 21, 113-130 (1929) andalso by Wolf in Jour. Elisha Mitchell Soc. 38,127-137 (1922).

In carrying out the present process a fungus of the class Basidiomycetesis prepared in aqueous suspension from agar slants or other sources, andadded to a prepared medium consisting of a source of carbon, nitrogenand mineral elements. The sources of carbon are well known to themicrobiologist and include corn starch, molasses, maltose, sucrose,glycerol and mannitol; various organic acids such as citric, malic,acetic; various natural products containing carbohydrates such as cornsteep liquor, soybean meal, cotton seed mealand many other availablematerials'which have been used heretofore as a source of carbon infermentation processes.

Usually a variety of the above can be used in the medium with goodresults.

Sources of nitrogen include some of the above enumerated materials suchas corn steep liquor, soybean meal, cotton seed meal and the like. Othersources can be beef extract, casein, yeast, enzymatically digestedproteins and degradation products including peptones, amino acids andother proteinaceous materials. Various inorganic sources of nitrogenincluding ammonium salts, nitrates and the like may also be used as asource of 'assimilable nitrogen to provide a favorable growth substratefor fungi of the Basidiomycetes class.

13,488) (D-10), Polyporus Often, much of the mineral requirements of thefermentation are present in the crude materials that are used to furnisha source of carbon and nitrogen or occur Usually, how- 2,982,695 fatented May 2, 1901 ICC of the fungus. Cations and anions are desirablein added amounts including phosphate, sulfate, chloride, sodium,potassium, magnesium, iron, calcium, cobalt, manganese and variousothers. Since the use of mineral elements in supporting the growth offungi is well understood, elaboration appears to be unnecessary.

Steroids of the pregnene series useful in the process of the presentinvention are Reichsteins Substance S, 21-hydroxy progesterone and thelike.

The fungi of the Basidiomycetes class grow at all temperaturesbetween 10and35 C. and it is possible to carry out the oxidation process withinthese ranges. Temperatures between 15? and 30 C. are preferred. Thereaction usually is carried out at about 28 C.

During the fermentation process, aeration is-provided by forcing sterileair through the medium. Mechanical agitation is also used to keep themycelium and other insoluble materials in suspension. Antifoaming agentssuch as silicone, glycerides, oils and the like may be added from timeto time in the amounts needed.

In carrying out the process of the present invention the fungus ispreferably grown in a suitable medium such as described hereafter in theexamples until suflicient growth is exhibited. This period is usuallyabout two days. The pregnene to be oxidized is then added to thefermentation medium in solution, it having been previously dissolved inan alcohol or other solvent which will not adversely effect thefermentation.

Following the completion of the reaction which can be determined bypaper chromatographic analysis, the mycelium is removed and the filtrateextracted with a solvent such as methylene dichloride, chloroform, orethyl acetate. The desired products can then be obtained by removalofthe solvent and purification in a well known manner.

In large scale fermentations, the crude product or products may berecovered from the fermentation beer by simple solvent extraction usinga suitable waterimmiscible solvent such as chlorinated lowerhydrocarbons, esters, ketones and the like. Further purification andseparation of the steroid products fro'in the extract may beaccomplished by well known methods. Separation of steroid mixtures oftenrequire the use of chromatography. 7 The compounds of the presentinvention have glucocorticoid activity and therefore are useful in thetreatment of arthritis, bursitis and other collagen diseases.

The following specific examples illustrate in detail themonohydroxylation of steroids of the pregnene series.

Example 1 An aqueous suspension of growth from a test tube agar plant ofPolyporus tulipiferus is divided into 6 portions as inoculum for 6-500n11. flasks, each of the latter containing ml. of a medium composed of0.22% soybean meal, 0.3% corn steep liquor, 1.0% glucose, 0.25% yeastextract, 0.3% ammonium dihydrogen phosphate and 0.25% calcium carbonate(pH adjusted to 7.0 with sodium hydroxide). The six flasks are placed ona reciprocating shaker at 2628 C. until sufficient growth (usually 2days) is exhibited. At this point flasks 1 and 2, 3 and 4, and 5 and 6,respectively, are combined and used as inoculum, for three bottles eachcontaining 12 liters of a medium consisting of 5%. dextrose, 2.0% cornsteep liquor and 0.2% K HBO The three bottles designated 1, 2, and 3 arekept at 26;;28" C. until the aerated, mechanically stirred growth issatis factory. At this time the substrate is added as a methanolsolution and the fermentation allowed to continue paper stripchromatography shows the substrate to be .3 exhausted. The three bottlesare then. harvested, the contents pooled and used for isolation studies.

4 Example 4 The same fermentation conditions are used as described forExample 3, except that 2l-hydroxyprogesterone is Temp., Prefersem r usedas steroid substrate instead of Compound S. Bottle O., of mentatationTotal Substrate Fermentiontimc time, Time tation Culture R; values inSolvent System XII 1 26-28 96 46 142 0.2 mg. of Substance (D-G) Polypoms cinna- Sper ml. medium. barinus "-0.44 0.65v 0.80 0.85 2; 26-28 4822 70 D0. (D-) Polyporus tulipiv 2H8 22 (dither-12.2555; aria S' ii 3'23393 "6'55 T Products in l Y Substame The polarity of the product at R;0.44 shows that the .55' Y Q Y Substance S and lsts'hydmxy Substance 15product can be l53,2l-dihydroxyprogesterone and that65,2l-dihydroxyprogesterone and 14a,21-dihydroxypro- Example 2 gesteroneboth are likewise in the area of R, 0.60-0.65 in Thirty-six liters offermentation beer prepared as in the solvent system used. Example 1 isfiltered to remove the mycelia, and the filtrate is extracted twice withequal volumes of ethyl Example? acetate. The ethyl acetate extracts arepooled and washed A test tube P 0f F 00608 18 W ed i h with their volumeof a 2% sodium bicarbonate soluof a S ter11 e Sal-me P one ml of theresult" tion, and the washed extract is concentrated under remgsljsPenslon used to W test tube duced pressure. The concentrated extractis dried with contammg 1O of a medmm conslstmg of 20% sodium sulfate,treated with 2 g. of activated carbon, 3 9 sodlumfhlo'nde and-20% Y,comsteep and filtered. The filtrate is concentrated to a residue andThe tube 15 Placed jeclpmcatmg Shaker chromatographed on a 1 kg.diatomaceous earth column at for 95\h0}1r$- At l P 2 gof 1161611- usinga water:dioxanezcyclohexane system (1:5 :4). The Stems SubStaPce S addedm 9' of ethanol .peak fractions of the 14u-hydroxy,6e-hydroxy, and 15phee mentation allowed to continue for 72 hours. At hydroxy derivatives, asshown by absorbflncy m th s time the tube 1s harvested and the mashextracted Hated A, B and C and appgaring at 1.0, 215, and 39 tw1ce W1thdouble volumes of methylene dichloride. The respectively), arecollected, and each is concem combined extracts are concentrated andthen sub ected treated to dryness, taken up into 150 ml. of'hot ethylPaper chromatfgraphyg ,T' chmmatogrfm acetate, treated with 1 g. ofactivated carbon, and filtered. three prfducts Wlth mobllmes conslstent'wlth the The twee filtrates (A, B, and C) are each concentrated 35terpretation that they are the 6;9,l4a, and lS/i-monohyto about 25 ml.and chilled. B and C readily crystallize "droxyl denvatlves of Substanceout and are removed by centrifugation and recrystallized E l 5 from hotmethanol, yielding 178 mg. of B and 162 mg. 'of C. The A material,because of its greater solubility, 4 tube g of q COCOS is washed wlth'is taken to dryness and crystallized from methanol-ether, S z g i gg gg$223 z f gfi fg yielding 61 mg. All three substances analyze fortrihydroxYPregnenef 10 t of a P conslstmg of 20% diones. A is14a-hydroxy S (14a,17o:,21-trihydroxy-4- 9 g chlQIldB and 0% (w/v.) cornsteep preguene-3,20-dione). Its infrared curve is identical with lquor;e m e was placed upon. a reflpmcatmg Shaker that of an authentic sample.B is 6B-hydr0xy S (65,1711, at 28 for 96 q At.th1s pomt 2 of 2l tlihydroxy 4 pregnene 320 dione) as shown by agree, hydroxyprogesterone isadded in 0.2 ml. of ethanol and ment of physical properties with thoserecorded in the 3 to cgntmue g 72 r At literature, particularlycharacteristic are the UV. absorpg i iigss g i g z 3: :32? ggfifi gf ifiggsg ggaziggggggg 33E133 gg gg i The combined extracts areconcentrated and then subby the fact that its infrared spectrum isidentical with listed paper chromatography .cilmmatofgram canasserts:artistes 1ZZ EEZ 1%%$iZS a. 3 Example 3 giggggxyl derivatives ofZl-hydroxyprogesterone are pro- About (SS-1.0 in]. of a 5 aqueoussuspension of W glaim; spores an y 111m scrape Tom a test e agar 1. Arecess for the 6 ,l4ot and 15 -monoh d X slant of Cariolua versicolor(Lederle 13-10) are inocuti of 2 1-h d 3,zo-dfketo ye n g hi h301333;;lated in 100 In f steriliZed medium (5 dextrose, subjecting saidpregnenes to the fermentative enzymatic m P liquor, and 01% 2 4) m a 500m 60 action of a fungus of the group consisting of Coriolus fi Thel'noclllated flasksa are Placed 011 feclpl'ocatlng versicolor, Polyporustulipiferus, Polyporus 'cinnabarinus shalce; ans incubaed atditidC2.0for 66dh0ur1s. dAftei' thrls d Pam; o .P S11 5 81168 15 a e g 1580Ve 111 m 2. A process for the 6 ,l4a,15 -monoh drox lation of of 70%ethyl alcohol per flask. Samples for chroma-170,21-dihydroxy-4-pregnine-3,20 dione w hich Zomprises :t }g1;1pl3falssay are 1taken at one daydinterlvalzsg 101 subjecting said pregneneto the fermentative enzymatic o ro pus myceium are extracte wit m. 0action of Pol orus tuli ierus. ethyl acetate by shaking the mixture for5 minutes. After 3. A proceg s for the 5514a,lSfi-monohydroxylation ofstanding to separate the layers the ethyl acetate extract17et,21-dihydroxy-4-pregnene-3,20-dione which comprises is transferredto a conical centrifuge tube and the solvent subjecting said pregneue tothe fermentative enzymatic evaporated in a warm water bath with the aidof an air action of Coriolus versicolor. jet. The dried residue issubmitted to qualitative paper 4. A process for the65,1411,15,8-monohydroxylation of stnp chromatographic assay and givesevidence of 3 170:,21-dihydroxy-4-pregnene-3,20-dione which comprisesdefinite chromatogram spots with R, values of 0.23, subjecting saidpregnene tothe fermentative enzymatic 0.36, and 0.63 in solvent systemIXA indicative of 1518- action of Poria cocos.

hydroxy S, oe-hydroxy S, and l ta hydroxy 5, respectively. 5. A. processfor the 6,8,14a,l5,8 monohydroxylation of 21-hydroxy-progesterone whichcomprises subjecting 21-hydroxyprogesterone to the fermentativeenzymatic action of Polyporus oinnabarinus.

6. A process for the 6p,14a,ISfi-monohydroxylation of21-hydroxy-progesterone which comprises subjecting21-hydroxyprogesterone to the fermentative enzymatic action of Polyporustulipiferus.

7. A process for the 618,140,ISB-monohydroxyiation of21-hydroxy-progesterone I which comprises subjecting21-hydroxyprogesterone to the fermentative enzymatic action of Poriacocos.

8. A process for the 63,140; and 15B monohydroxylation of21-hydroxy-3,ZO-diketopregnenes which comprises subjecting saidpregnenes to the fermentative enzymatic action of COIiiollls versicolor.

9. A process for the 63,140; and 15p monohydroxylation of21-hydroxy-3,ZO-diketopregnenes which comprises subjecting saidpregnenes to the fermentative enzymatic action of Polyporus tulipiferus.

10; A process for the 613,140: and 15/3 monohydroxylation of21-hydroxy-3,ZO-diketopregnenes which comprises subjecting saidpregnenes to the fermentative enzymatic action of Polyporuscinnabarinus.

11. A process for the 65,140: and 155 monohydroxylation of21-hydroxy-3,20-diketopregr1enes which comprises subjecting saidpregnenes to the fermentative enzymatic action of Poria cocos.

No references cited.

1. A PROCESS FOR THE 6B,14A AND 15B-MONOHYDROXYLATION OF21-HYDROXY-3,20-DIKETOPREGNENS WHICH COMPRISES SUBJECTING SAID PREGNENESTO THE FERMENTATIVE ENZYMATIC ACTION OF A FUNGUS OF THE GROUP CONSISTINGOF CORIOLUS VERSICOLOR, POLYPORUS TULIPIFERUS, POLYPORUS CINNABARINUSAND PORIA COCOS.